Reduced dose intensity of docetaxel plus capecitabine as second-line palliative chemotherapy in patients with metastatic gastric cancer: a phase II study.

BACKGROUND: A phase II study was conducted to evaluate the efficacy and safety of a combination regimen of a reduced dose intensity of docetaxel (Taxotere) plus capecitabine in pretreated patients with metastatic gastric cancer. PATIENTS AND METHODS: Twenty-eight patients with documented progression on or within 3 months of a cisplatin-based chemotherapy were enrolled between April 2004 and November 2006. Docetaxel (60 mg/m2 on day 1) plus capecitabine (1000 mg/m2 twice daily on days 1-14) were given every 3 weeks. RESULTS: All patients were assessable for safety and 25 (89%) for tumor response. Median age was 63 years, and median follow-up was 13.3 months. Overall response rate was 29% (95% confidence interval 11% to 46%), while an additional 36% had stable disease. The median time to progression and median overall survival was 4 and 6 months, respectively. The most common clinical adverse events (all grades) were neutropenia (78%), hand foot syndrome (HFS) (53%), fatigue and alopecia (50%) and diarrhea (43%). However, with the exception of grade 3-4 neutropenia, which was seen in 36% of patients, other severe adverse events were rare. There were no treatment-related deaths. Treatment delays or dose reductions were necessary in 18 out of 104 cycles. CONCLUSIONS: A reduced dose intensity of docetaxel plus capecitabine is a valuable regimen for second-line treatment in this setting of patients. This approach warrants further investigation as a promising chemotherapy option for chemonaive patients with metastatic gastric cancer.

Lapatinib in breast cancer.

Aberrant activation of some members of human epidermal growth factor receptor (HER) family plays a key role in breast carcinogenesis. Lapatinib is an oral dual tyrosine kinase inhibitor selective for inhibition of epidermal growth factor receptor (EGFR/ErbB1) and HER2/ErbB2. Having more targets, probably its antitumor activity could be more efficient. Clinical data have shown that lapatinib is active in HER2-positive breast cancer as monotherapy, in combination with trastuzumab, and in trastuzumab-resistant patients. Phase I clinical trials have shown also that lapatinib is well tolerated, with mild diarrhea and skin rush as common toxic effects and low incidence of cardiotoxicity. Phase II and III clinical trials' data provide encouraging evidence of the clinical effectiveness of lapatinib in advanced or metastatic breast cancer and for its potential in patients with brain metastases. Interim results from the large, phase III trial in 392 patients showed that in combination with capecitabine lapatinib almost doubled time to progression when compared with capecitabine alone. Several clinical trials that explore the efficacy of lapatinib in combination with conventional chemotherapeutic agents [paclitaxel (Taxol), capecitabine and platinoids], hormonotherapy and other target therapies are ongoing in advanced breast cancer or in neo-adjuvant and adjuvant settings. Our improved understanding of the biology of breast cancer and the use of biomarkers for identification of specific subtypes are allowing us to bring patient-specific novel therapies such as lapatinib to the clinic.

Rectal neurogenic sarcoma: case report and review of the literature.

A neurogenic sarcoma without NF-1 was discovered in a 73-year-old woman in the anorectal region, an unusual site for these tumors. The tumor was of high-grade malignancy and deeply located with mesorectal infiltration; it did not originate from a major nerve. We presume an origin from less differentiated neural crest cells and present a review of the literature on the best treatment for these neoplasms.

Overexpression of multidrug resistance-associated p170-glycoprotein in acute non-lymphocytic leukemia.

Resistance to several cytotoxic agents, including anthracyclines, vinca alkaloids and epipodophylline derivatives (multidrug resistance, or MDR) can develop in tumor cells by overexpression of a 170-kd glycoprotein (p170) which is an essential component of a membrane transport system leading to increased drug efflux and decreased intracellular drug concentration. By means of a p170-directed monoclonal antibody (MRK-16) and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique), we investigated the expression of p170 in marrow blast cells of 59 cases (38 at diagnosis and 21 in relapse) of acute-non-lymphocytic leukemia (ANLL). The proportion of strongly MDR-positive cells was higher in relapse that at diagnosis (median 15.5% vs 1.5%). Out of 31 patients who were evaluable for the results of first remission induction, failure of first-line treatment (including Daunorubicin, standard-dose and high-dose Arabinosyl Cytosine, and sometimes also Mitoxantrone) occurred in 8/22 MDR-positive cases and in 1/9 MDR-negative ones (p = 0.21). Failure of first-line treatment was always associated with a progressive increase of p170 expression. Total failures (no remission plus early relapse) were more frequent (p = 0.001) among MDR-positive cases (16/22) than among the others (2/9). These data show that MDR is very frequent in ANLL also at diagnosis and suggest that MDR can contribute to early failure of standard treatment.

Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

Bone marrow purging by a xanthine oxidase-antibody conjugate.

The selective cytotoxicity of the xanthine oxidase conjugated to an 8A monoclonal antibody recognizing a human plasma cell-associated antigen has been described. The selectivity and the toxicity of the hypoxanthine/conjugated xanthine oxidase system was increased by removing the excess of conjugate and by adding chelated iron. Under these experimental conditions the cytotoxicity of the conjugate exceeded that of free xanthine oxidase by one order of magnitude. The conjugate effectively purged bone marrow from infiltrating neoplastic plasma cells and added target Raji cells, provided blood was removed and bone marrow peroxidases were exhausted. In conditions of purging effectiveness the conjugate had no toxicity to CFU-GM. No toxicity to mice was observed after i.v. injection of xanthine oxidase-antibody conjugate up to 2.9 U/kg body weight. Thus the hypoxanthine/conjugated xanthine oxidase system could be an effective and nontoxic tool for the ex vivo bone marrow purging in multiple myeloma patients for autologous transplantation.

Evaluation of LAK-mediated tumor cell killing in a plasma clot clonogenic assay.

An assay based on the inhibition of the cloning capacity in a plasma clot semisolid medium assay has been used to test the sensitivity of the Raji cell line to lymphokine-activated killer (LAK) cells. This method overcomes some limitations intrinsic to the widely employed 51Cr release assay and always shows a higher degree of sensitivity. No inhibition of colony growth was found when the effector cells were plated without prior pre-incubation with interleukin 2 or with the addition of the medium derived from the LAK cells. Though more time-consuming than the classic 51Cr release assay, this technique does not require radioactive material. This test may be suitable for a more precise evaluation of LAK activity and for the study of the mechanisms involved in cell killing.

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.

Cryopreserved autologous bone marrow transplantation in patients with acute nonlymphoid leukemia: chemotherapy before harvesting is the main factor in delaying hematological recovery.

We analyzed the kinetics of hematological recovery after autologous bone marrow transplantation in 13 patients with acute nonlymphoid leukemias (ANLL). A comparison was made with 31 patients with non-Hodgkin's lymphoma (NHL) whose bone marrow was harvested and cryopreserved, either at diagnosis or after intensive chemotherapy. Hematological recovery of ANLL patients was similar to that of pretreated NHL patients and significantly slower than that of untreated NHL patients. We suggest that chemotherapy before harvest (more than the possible decreased stem cell marrow potentiality resulting from the underlying disease) appears to be the main factor responsible for delayed recovery after autologous bone marrow transplantation in ANLL.

Improved methods for the determination of 5-bromo-2-deoxyuridine labelling index in clones and colonies growing in plasma clots.

Bromodeoxyuridine (BrdUrd), an analogue of thymidine is one of the most employed marker to detect the S-phase of the cell cycle. Difficulties are described for the in situ detection of S-phase cells in normal and neoplastic growing clones. In this paper we propose new methods for the detection of BrdUrd in neoplastic clones, growing in plasma clots. In particular, these methods are based on the immunocytochemical staining with horseradish peroxidase and alkaline phosphatase, as well as on a new immunofluorescent streptavidin technique. They allow easy detection of S-phase cells with an inverted light microscope.

Mixed lymphocyte reactions evaluated by means of bromodeoxyuridine incorporation.

The mixed lymphocyte reactions are usually performed by the uptake of 3H thymidine (3H TdR) in the study of histocompatibility for allogeneic bone marrow transplantation. Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be used as an alternative marker of proliferation. In this study we compared the evaluation of the proliferative activity of alloreactive lymphocytes in MLR by 3H TdR and BrdUrd incorporation in flow cytometry. The results show that BrdUrd is able to recognize proliferative activity at least 24-48 hours before 3H TdR, and allows discrimination of proliferation of a few cells despite its proximity to the autologous controls. However, the large quantity of cells needed for every combination is presently a disadvantage of the method.

Comparison of the DNA Content, Bromodeoxyuridine Incorporation and Ki-67 Antigen Expression in Human Acute Myeloid Leukemia.

The cell kinetics of twenty-two acute myeloid leukemias (AML) were investigated by means of flow cytometry evaluating the S-phase DNA content, bromodeoxyuridine labelling index (BrdUrd L.I.) and Ki-67 antigen expression. Eight patients showed a good correlation between the DNA content and BrdUrd L.I., while nine gave rise to divergent results. In the remaining five patients the S-phase DNA content could not be evaluated due to the presence of an additional aneuploid population. The Ki-67 antigen expression defined the extent of the growth fraction in all cases and allowed for better characterization of the cell cycle. These results suggest that the three methods explore only partly overlapping events; thus, it seems that a reliable picture of the cell kinetics in leukemic populations can only be achieved by combining all these methods.

Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.

Evidence that long-term bone marrow culture of patients with multiple myeloma favors normal hemopoietic proliferation.

Long-term bone marrow cultures (LTBMC) were initiated with marrow aspirate cells from 12 patients with multiple myeloma (MM) using the Dexter system. The myeloid and the neoplastic myeloma cell growths were evaluated for up to 6-9 weeks. Our results demonstrate the development of an adherent layer capable of supporting normal granulopoiesis with a concomitant drop in the growth of myeloma cells. The B lymphocyte monoclonal proliferative compartment was also studied with bromodeoxyuridine (Brdurd), an analog of thymidine incorporated during the S-phase, and the labeling index was calculated. The ability to form myeloma stem cell colonies in a modified plasma clot short-term assay was also evaluated. The results confirmed that the neoplastic B lineage compartment was not able to grow in Dexter's system for more than 4 weeks in 11 of 12 cases studied, with the disappearance of Brdurd-positive cells after two weeks, whereas LTBMC were able to sustain the growth of myeloid progenitors. These data indicate the potential applicability of this culture method in selecting normal hematopoietic progenitors from patients with multiple myeloma. This approach can have significant implications for aggressive treatment of patients with multiple myeloma, especially in trials involving autologous bone marrow transplantation (ABMT).

Selective killing of CD4+ and CD8+ cells with immunotoxins containing saporin.

Saporin, a ribosome-inactivating protein from the seeds of Saponaria officinalis, was covalently linked to an anti-CD4 monoclonal antibody. The resulting immunotoxin at 10(-9)M concentration was toxic to CD4+ lymphocytes without affecting other cells. Selective elimination of CD4+ and CD8+ cells was also obtained with murine monoclonal anti-CD4 and anti-CD8 antibodies and an immunotoxin consisting of saporin linked to an anti-mouse IgG antibody.

An immunotoxin containing momordin suitable for bone marrow purging in multiple myeloma patients.

Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.

Normal myeloid progenitors (CFU-GM) in multiple myeloma: a preliminary study in view of autologous BMT.

Normal granulocyte-macrophage precursors (CFU-GM) were studied in 65 multiple myeloma patients by means of culture assays. The patients were divided into separate groups on the basis of previous therapy (i.e. analysis performed at diagnosis or after chemotherapy), time elapsed from the last therapy (i.e. more or less than 1 month) and clinical features of the disease (i.e. tumor stage, immunoglobulin type, bone marrow plasma cell infiltration). The results were evaluated by Wilcoxon rank sum test and linear regression analysis. There was no statistical difference in CFU-GM cloning efficiency or in the number of CFU-GM/ml of bone marrow, even though a larger CFU-GM recovery was found in patients evaluated at diagnosis or at least 1 month or more from previous chemotherapy. In addition, no correlation was demonstrated between bone marrow plasma cell percentage and CFU-GM cloning efficiency. This finding was confirmed by the number of myeloid bone marrow cells in S-phase, assessed by the bromodeoxyuridine labeling index, which showed similar results in patients with different degrees of plasma cell infiltration. In conclusion our data indicate that the granular-monocytic lineage keeps its cell-line potentiality regardless of the degree of marrow plasma cell infiltration and the type of therapeutic approach. These data suggest that autologous bone marrow transplantation might be feasible even in patients with a large neoplastic infiltration.

Targeting of a plasma cell line with a conjugate containing xanthine oxidase and the monoclonal antibody 62B1.

We report on the preparation of an antibody-conjugated enzyme consisting of xanthine oxidase, a free-radical-producing enzyme, linked to the 62B1 monoclonal antibody, which recognizes the last steps of differentiation of B cell lineage (plasma cell and hairy cells). The conjugate specifically kills target cells, retaining both enzymic and immunological properties, without any damage to normal myeloid clonogenic efficiency. The model is suitable for ex vivo bone marrow purging in multiple myeloma patients.

Immunotoxins containing saporin 6 and monoclonal antibodies recognizing plasma cell-associated antigens: effects on target cells and on normal myeloid precursors (CFU-GM).

Monoclonal antibodies 8A and 62B1, recognizing plasma cell-associated antigens, were covalently linked to saporin 6, a ribosome-inactivating protein similar to the A-chain of ricin. Both immunotoxins were tested on target human cell lines U266 and Raji, on non-target K562 cell line and on myeloid CFU-GM progenitors. The cloning efficiency and viability of target cells were strongly reduced by 8A-saporin 6 and 62B1-saporin 6 immunotoxins, with an ID50 up to 200,000-fold lower than free saporin 6, whilst the K562 non-target cell line was unaffected. Normal human myeloid precursors (CFU-GM) were inhibited by immunotoxins only to a limited extent. An application of this model for autologous bone marrow transplantation in multiple myeloma patients is proposed. Since no eradication of cloning target cells was achieved by a single immunotoxin, mixtures made with different antibodies could help to reach this goal.